Homogeneous engineered phage populations

ABSTRACT

Provided are engineered phages populations, which are homogeneous in length, as well as methods of making and methods of using such phages. Also provided are engineered chlorotoxin-phages as well as their methods of making and using. The disclosed homogeneous phage populations and chlorotoxin-phages may be used, for example, for treating and/or imaging tumors, such as central nervous system tumors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.16/040,793, filed Jul. 20, 2018, which claims priority from U.S.Provisional Application No. 62/535,604, filed Jul. 21, 2017, the entirecontents of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. P30CA014051 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-WEB and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 19, 2021, isnamed sequence.txt and is 2,432 bytes.

FIELD

The present application relates generally to the field of biotechnologyand more specifically to engineered bacteriophage systems and methods ofmaking and using them.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 19, 2018, isnamed sequence.txt and is 3 KB in size.

SUMMARY

One embodiment is a method of making a homogeneous phage populationand/or a homogeneous ssDNA population, comprising: obtaining a firstartificial plasmid comprising an f1 origin replication sequence;obtaining a second artificial plasmid that does not comprise an f1origin replication sequence; and co-transforming the first artificialplasmid and the second artificial plasmid into a bacterial strain toproduce a homogeneous phage population and/or a homogeneous ssDNApopulation, wherein the first and second artificial plasmid togethercontain sequences encoding a complete library of phage coat and assemblyproteins.

Another embodiment is a homogeneous engineered phage population, whereinat least 30% of phages in the phage population have a length within 15%of a length value, which is selected from 10 nm to 10 microns.

Yet another embodiment is a homogeneous engineered phage population,wherein at least 30% of phages in the phage population have a lengthwithin 8 nm of a length value, which is selected from 10 nm to 10microns.

Yet another embodiment is a phage comprising chlorotoxin expressed by acoat protein of the phage.

Yet another embodiment is a kit for making a homogeneous phagepopulation and/or a homogeneous ssDNA population comprising: a firstartificial plasmid comprising an f1 origin replication sequence; and asecond artificial plasmid, which does not comprise an f1 originreplication sequence, wherein the first and second artificial plasmidtogether contain sequences encoding a complete library of phage coat andassembly proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1 is an Inho construct plasmid map, with site of circular ssDNAproduction region shown in dark green.

FIG. 2 schematically illustrates phage-inho production: inho plasmidswhen co-transformed with another plasmid (RM13-f1) coding for phageproteins produce short phage that packages the inho genomes, whichcontrol the length of the phage.

FIG. 3A-C are atomic force microscopy (AFM) images of inho 1960, inho475 and inho 285 phages. FIG. 3D is a plot showing a measured length ofphage in nm as a function of packaged ssDNA size.

FIG. 4 shows a nucleotide sequence (SEQ ID No. 1) for M13 code modifiedwith chlorotoxin sequence at the p3 gene.

FIG. 5 shows an AFM image of chlorotoxin-phage with negatively chargedgold particle interaction at p3.

FIG. 6A-D show fluorescent images for internalization of CTX Phage inAdult Human U87MG Glioblastoma and Pediatric Human D458 MedulloblastomaCells. In vitro cellular uptake and colocalization of CTX phage (red) inthe Golgi apparatus (green) in (A & B) human U87MG glioblastoma and (C &D) human D458 medulloblastoma cells. Nuclei shown in blue. (scale bar=5μm).

FIG. 7 presents results of Intravital Multiphoton Imaging Showing TumorUptake of CTX-Phage in the Brain of a NCR Nude Mice with an OrthotopicHuman U87MG Glioma Xenograft.

FIG. 8 presents results of Intravital Multiphoton Imaging Showing TumorUptake of CTX Phage in the Brain of a C57/BL6 Mouse With an OrthotopicSynergic Mouse GL261 Glioma Xenograft.

FIG. 9 relates to the use of CTX phage (long and short) with NIRIIimaging of glioma (ex-vivo, post IV injection).

FIG. 10 schematically illustrates small phage model.

FIG. 11 schematically illustrates passage of small CTX phage across theBBB.

FIG. 12 schematically illustrates multi-phage scaffolds &nanostructures.

FIG. 13 is a map of CTX insertion.

FIG. 14 shows a replication sequence from M13-ori (SEQ ID No. 2).

FIG. 15 is a map of RM13-f1, M13-f1with p8 modification (DDAH), with p3modification (HIS6 (SEQ ID NO: 7)), with p8 and p3 modification (DSPH &CTX).

FIG. 16 shows sequences of f1 replication modified for origin only (SEQID No. 3), termination only (SEQ ID No. 4 and SEQ ID No. 5), and thepackaging signal as found in M13-ori (SEQ ID No. 6).

FIG. 17 schematically depicts ssDNA sequence site of inho construct.

FIG. 18 schematically illustrates examples of inho sequence insertionsfor modified ssDNA length.

FIG. 19 schematically illustrates examples of inho ssDNA productionsequence combined with RM13-f1protein sequences: with differing plasmidreplication origins (p15Ori and pUC Ori) for varying yield effectsduring E. coli growth.

FIG. 20 presents results of analysis of packaged genomes: TBE-PAGE gelof the inho 285, 311, 344, 475, 1310, 1960 constructs, stained withSYBR-Gold. Inho ssDNA run higher than the reference ruler (designed fordsDNA).

FIG. 21 presents results of analysis of p3 coat protein: anti-p3 WesternBlot from NU-PAGE gel run of inho, CTX, and Wildtype phage.

FIG. 22 shows a histogram distribution of inho phage sizes as measuredfrom AFM imaging: for inho475 and inho1960 phages, about 90% of allmeasured lengths fall within the base size bin.

FIG. 23 provides a list of M13 bacteriophage proteins.

DETAILED DESCRIPTION

Unless otherwise specified, “a” or “an” refers to one or more.

The present inventors developed a method of producing homogeneous phagepopulations. The method may be applied to a phage with an isolatablepackaging signal sequence. In some embodiments, a phage may be afilamentous phage. Examples of filamentous phages include Ff phages,such as M13 bacteriophage, Ike phage, f1 phage and fd phage. Yet inother embodiments, a phage may be a non-filamentous phage, such aslambda phage or adeno-associated virus.

The method includes obtaining two artificial, i.e. man-made, phageplasmids and co-transforming the two artificial plasmids into acompetent bacterial strain to produce a homogeneous phage population. Insome embodiments, the product of the co-transformation may be amplifiedto produce the homogeneous phage population.

For Ff phages, such as M13 bacteriophage, the bacterial strain may beone that carries the F-episome. In some embodiments, the bacterialstrain may be a strain of E. coli. For example, the bacterial strain maybe DH5α, NovaBlue, XL10, ER2738 or XL1. In some embodiments, thebacterial strain may be an antibiotic resistant strain, such as atetracycline resistant bacterial strain.

The two artificial plasmids are preferably such that together theycontain sequences for a complete library for coat and assembly proteinsfor a particular type of phage. In other words, the two artificialplasmids contain together at least one copy of each protein encodinggene present in a corresponding wildtype phage genome. For example, forM13 phage, the two artificial plasmids will contain together sequencesfor each of pI-pXI proteins, see FIG. 23.

The first of the artificial plasmids may be a modified (compared to thewildtype) circular double strand (ds) DNA phage plasmid. The firstartificial plasmid may contain an f1 origin replication sequence. Thefirst artificial plasmid may prepared by modifying a wildtype phageplasmid in certain areas. For example, at least 2 base pairs or at least5 base pairs or at least 10 base pairs or at least 20 base pairs or atleast 50 base pairs or at least 100 base pairs or at least 150 basepairs or at least 200 base pairs or at least 250 base pairs or at least300 base pairs or at least 350 base pairs or at least 400 base pairs orat least 450 base pairs or at least 500 base pairs or at least 600 basepairs or at least 700 base pairs or at least 800 base pairs or at least900 base pairs or at least 1000 base pairs or at least 1500 base pairsor at least 2000 base pairs or at least 2500 base pairs or at least 3000base pairs or at least 3500 base pairs or at least 4000 base pairs or atleast 4500 base pairs or at least 5000 base pairs or at least 5500 basepairs or at least 6000 base pairs may be added, replaced, and/orremoved. The modification(s) of the dsDNA acting as the first plasmidmay be used to control or define a length of phages in the phagepopulation and/or a length of produced ssDNA. In some embodiments, thef1 origin replication sequence may also function as an f1 terminationreplication sequence. Such sequence is illustrated, for example, in FIG.14. In some embodiments, the first artificial plasmid may also containat least one of an f1 termination replication sequence and a packagingsignal sequence, such as SEQ ID No. 6. In some embodiments, the firstartificial plasmid may contain an f1 termination replication sequence inaddition to the f1 origin replication. In such case, the ssDNA'sproducing section between the f1 origin replication sequence and the f1termination of replication sequence may be modified. For example, one ormore base pairs may be added, replaced or removed. In certainembodiments, at least 2 base pairs or at least 5 base pairs or at least10 base pairs or at least 20 base pairs or at least 50 base pairs or atleast 100 base pairs or at least 150 base pairs or at least 200 basepairs or at least 250 base pairs or at least 300 base pairs or at least350 base pairs or at least 400 base pairs or at least 450 base pairs orat least 500 base pairs or at least 600 base pairs or at least 700 basepairs or at least 800 base pairs or at least 900 base pairs or at least1000 base pairs or at least 1500 base pairs or at least 2000 base pairsor at least 2500 base pairs or at least 3000 base pairs or at least 3500base pairs or at least 4000 base pairs or at least 4500 base pairs or atleast 5000 base pairs or at least 5500 base pairs or at least 6000 basepairs may be added, replaced or removed. In some embodiments, the firstartificial plasmid may contain an f1 origin replication sequence, apackaging signal sequence and an f1 termination of replication sequence.The packaging signal sequence may comprise SEQ ID NO. 6 (FIG. 16) A)ssDNA producing section between the f1 origin replication sequence andthe packaging signal sequence and/or B) ssDNA producing section betweenthe packaging signal sequence and the f1 termination of replicationsequence may be modified. For example, one or more bases in one or bothof these sections may be added, replaced or removed. In certainembodiments, at least 2 base pairs or at least 5 base pairs or at least10 base pairs or at least 20 base pairs or at least 50 base pairs or atleast 100 base pairs or at least 150 base pairs or at least 200 basepairs or at least 250 base pairs or at least 300 base pairs or at least350 base pairs or at least 400 base pairs or at least 450 base pairs orat least 500 base pairs or at least 600 base pairs or at least 700 basepairs or at least 800 base pairs or at least 900 base pairs or at least1000 base pairs or at least 1500 base pairs or at least 2000 base pairsor at least 2500 base pairs or at least 3000 base pairs or at least 3500base pairs or at least 4000 base pairs or at least 4500 base pairs or atleast 5000 base pairs or at least 5500 base pairs or at least 6000 basepairs may be added, replaced or removed in one or both of thesesections.

In some embodiments, the first artificial plasmid may contain an f1origin replication sequence and a packaging signal sequence. Thepackaging signal sequence may comprise SEQ ID NO. 6 (FIG. 16) A) ssDNAproducing section between the end of the f1 origin replication sequenceand the packaging signal sequence and/or B) ssDNA producing sectionbetween the packaging signal sequence and the start of f1 origin ofreplication sequence may be modified. For example, one or more basepairs in one or both of these sections may be added, replaced orremoved. In certain embodiments, at least 2 base pairs or at least 5base pairs or at least 10 base pairs or at least 20 base pairs or atleast 50 base pairs or at least base pairs or at least 150 base pairs orat least 200 base pairs or at least 250 base pairs or at least 300 basepairs or at least 350 base pairs or at least 400 base pairs or at least450 base pairs or at least 500 base pairs or at least 600 base pairs orat least base pairs or at least 800 base pairs or at least 900 basepairs or at least 1000 base pairs or at least 1500 base pairs or atleast 2000 base pairs or at least 2500 base pairs or at least 3000 basepairs or at least 3500 base pairs or at least 4000 base pairs or atleast 4500 base pairs or at least 5000 base pairs or at least 5500 basepairs or at least 6000 base pairs may be added, replaced or removed inone or both of these sections.

The first artificial plasmid leads to the replication of a packageablecircular ssDNA, i.e. a plasmid, which may be packaged, after theco-transformation of the first and second artificial plasmids into abacterial strain.

In some embodiments, the first artificial plasmid may be modified insuch a way that it may be missing one or more phage protein genes. Insuch case, at least one copy of the phage protein missing gene(s) may bepresent in the second plasmid.

In some embodiments, the first artificial plasmid may be modified insuch a way that it may be missing all of phage protein genes. In suchcase, at least one copy of each phage protein gene may be present in thesecond plasmid.

In some embodiments, the f1 origin replication sequence may be amodified (compared to the wildtype one) f1 origin replication sequence.A non-limiting example of a modified f1 origin replication sequence ispresented in FIG. 16 as SEQ ID No. 3 (Dotto et al., PNAS, 1982, 79(23),7122-7126).

In some embodiments, the f1 termination of replication sequence may be amodified (compared to the wildtype one) f1 termination of replicationsequence. Non-limiting examples of modified f1 termination ofreplication sequences are presented in FIG. 16 and SEQ ID Nos. 4 and 5(Horiuchi, Genes to Cells, 1997, 2(7), 425-432).

In some embodiments, the first artificial plasmid may be missing apackaging signal sequence. Such sequence may be used to produce minimalsize phage populations and minimal size ssDNA. When the first artificialplasmid is missing a packaging signal sequence, it may be preferred touse the second artificial plasmid, which also misses a packaging signalsequence.

The second of the two artificial plasmids may be a helper phage plasmid,which is modified to disrupt the origin for packageable ssDNAreplication.

For example, the second artificial plasmid may be a helper phage plasmidwhich is modified so that it does not contain an f1 replication originsequence (either wildtype or modified). In some embodiments, the secondartificial plasmid may be a helper phage plasmid which is modified sothat it does not also contain a packaging signal sequence (eitherwildtype or modified).

In some embodiments, other than missing the f1 replication originsequence and optionally, the packaging signal sequence, the modifiedhelper phage plasmid used as the second artificial plasmid may includeall essential phage assembly components, i.e. contain sequences encodingall phage proteins (coat and assembly proteins). In such case, the firstplasmid may or may not be missing all these sequences.

The second artificial plasmid may be prepared, for example, from acommercial helper phage plasmid by reassembling the intergenic region todisrupt the origin for packageable ssDNA replication. For example, insome embodiments, a sequence comprising SEQ ID No. 2 may be removed froma helper phage plasmid. Examples of commercially available helper phageplasmids for M13 bacteriophage include M13KE , M13K07 and R 408.

The modified helper phage plasmid used as the second artificial plasmidmay include further modifications. For example, COLE1 (p15a-ori) plasmidreplication sequence may be included. Such inclusion may allow achievingan optimal copy numbers during bacterial growth, such as E. coli growth.The modified helper phage may also include one or more of additionalfunctionalization, such as adding chlorotoxin (CTX) sequence, such asSEQ ID No. 1, at the pIII gene; adding a sequence for DSPH or a sequencefor a carbon nanotube complexing peptide at the pVIII gene (Ghosh et al,PNAS 2014 111 (38) 13948-13953); adding a sequence encoding HIS-tag atthe pIII gene ((Hess et al, Bioconjug Chem., 2012, 23(7), 1478-1487);separating overlapping capsid sequences (Ghosh et al, ACS Synth Bio.,2012, 1(12), 576-582).

In some embodiments, the product of the co-transformation, which may bea bacterial colony comprising a phage population, may be amplified. Forexample, a bacterial growth media, such as a lysogeny broth media, maybe prepared. The bacterial colony, which may be the product of theco-transformation may be grown in the bacterial growth media to producea resultant bacterial culture. Then bacteria may be removed from theresultant bacterial culture to produce a supernatant containing a phageproduct. Such removal may be performed by centrifuging the resultantbacterial culture. The supernatant containing the phage product may beused for precipitating phage particles of the phage population. Thephage precipitated phage media may be centrifuged to pellet out thephage phage population. The pelleted phage population may be suspendedin a sterile solution, which may be, for example, a sterile buffer, orpurified water, such as MilliQ water.

In some embodiments, the product of the co-transformation may be storedfor up to three months prior to amplification. For example, the productof the co-transformation may be stored for at least 1 day up to threemonths or for at least 2 days up to three months or for at least 3 daysup to three months or for at least 5 days up to three months or for atleast a week up to three months or for at least 10 days up to threemonths or for at least two weeks up to three months or for at least 3weeks up to three months or for at least one month up to three months orfor at least 45 days up to three months and any values or subrangeswithin these ranges.

The present method may allow production of homogeneous phagepopulations. A phage population may be homogeneous when a substantialportion of phages of the population, e.g. at least 10% of thepopulation, has a phage length, which is substantially close to aselected phage length value. The selected length value may be determinedby selecting a particularly modified first artificial plasmid. Incertain embodiments, the selected length value may be determined byselecting a length (and/or a number of base pairs) of the firstartificial plasmid. In general, when all other conditions are the same,a longer (having more base pairs) first artificial plasmid will resultin a longer phage population, while a shorter (having less base pairs)first artificial plasmid will result in a shorter phage population. Forexample, artificial plasmids having 1960, 475 and 285 base pairs whenco-transformed with the RM13-f1second plasmid will give phages havinglengths of 280 nm, 100 nm and 50 nm.

In some embodiments, at least 10% of phages or at least 20% of phages orat least 30% of phages or at least 40% of phages or at least 50% ofphages or at least 60% of phages or at least 70% of phages or at least80% of phages or at least 90% of phages may be within 20% or within 15%or within 12% or within 10% or within 9% or within 8% or within 7% orwithin 6% or within 5% of the selected phage length value.

In some embodiments, at least 10% of phages or at least 20% of phages orat least 30% of phages or at least 40% of phages or at least 50% ofphages or at least 60% of phages or at least 70% of phages or at least80% of phages or at least 90% of phages may be within 20 nm or within 15nm or within 12 nm or within 10 nm or within nm or within 8 nm or within7 nm or within 6 nm or within 5 nm of the selected phage length value.

The selected phage length value may be, for example, a value between 10nm and 10000 nm (10 microns). For example, the selected phage lengthvalue may be from 10 nm to 8000 nm or from 10 nm to 6000 nm or from 10nm for 4000 nm or from 10 nm to 3000 nm or from 10 nm to 2000 nm or from10 nm to 1800 nm or from 10 nm to 1600 nm or from 10 nm to 1400 nm orfrom 15 nm to 8000 nm or from 15 nm to 6000 nm or from 15 nm for 4000 nmor from 15 nm to 3000 nm or from 15 nm to 2000 nm or from 15 nm to 1800nm or from 15 nm to 1600 nm or from 15 nm to 1400 nm or from 20 nm to8000 nm or from 20 nm to 6000 nm or from 20 nm for 4000 nm or from 20 nmto 3000 nm or from 20 nm to 2000 nm or from 20 nm to 1800 nm or from 20nm to 1600 nm or from 20 nm to 1400 nm.

In some embodiments, the selected phage length value may be below 50 nm.For example, the selected phage length value may be at least 10 nm butless than 50 nm or at least 10 nm but no more than 45 nm or at least 10nm but no more than 40 nm or at least 10 nm but no more than 35 nm or atleast 15 nm but less than 50 nm or at least 15 nm but no more than 45 nmor at least 15 nm but no more than 40 nm or at least 15 nm but no morethan 35 nm.

In some embodiments, the selected phage length value may be greater than50 nm. For example, the selected phage length value may be at least 55nm or at least 60 nm or at least 65 nm or at least 70 nm or at least 75nm or at least 80 nm or at least 80 nm or at least 85 nm or at least 90nm or at least 100 nm or least 110 nm or at least 120 nm.

In some embodiments, the selected phage length value may be greater than50 nm but less than a wildtype type phage length. For example, theselected phage length value may be greater than 50 nm but less than 880nm; or at least 55 nm but less than 880 nm or at least 60 nm but lessthan 880 nm or at least 70 nm but less than 880 nm or at least 80 nm butless than 880 nm or at least 100 nm but less 880 nm or greater than 50nm but less than 850 nm; or at least 55 nm but less than 850 nm or atleast 60 nm but less than 850 nm or at least 70 nm but less than 850 nmor at least 80 nm but less than 850 nm or at least 100 nm but less 850nm or greater than 50 nm but less than 820 nm; or at least 55 nm butless than 820 nm or at least 60 nm but less than 820 nm or at least 70nm but less than 820 nm or at least 80 nm but less than 820 nm or atleast 100 nm but less 820 nm.

In some embodiments, the selected phage length value may be greater thana wildtype type phage length. For example, the selected phage lengthvalue may be greater than 900 nm or at least 920 nm or at least 940 nmor at least 960 nm or at least 980 nm or at 1000 nm.

In some embodiments, the selected phage length may be from 10 nm to 150nm or from 10 nm to 120 nm or from 10 nm to 100 nm or from 10 nm to 40nm or from 60 nm to 120 nm. Phage populations having such lengths may bepreferred for imaging applications, when phages are used a biologicalprobe.

In some embodiments, the selected phage length value may be from 100 nmto 10 microns or from 100 nm to 5 microns or from 100 nm to 2 microns orfrom 100 nm to 1 micron or from 100 nm to 800 nm or from 950 nm to 10microns or from 1 micron to 10 microns. Phage populations having suchlengths may be preferred when phages are used in implant applications,such as hydrogel and/or scaffold applications.

The produced homogeneous phage population may have a phage count of atleast 1e12 pfu or at least 3e12 pfu or at least 5e12 pfu or at least1e13 pfu or at least 3e13 pfu or at least 5e13 pfu or at least 1e14 pfuor at least 3e14 pfu or at least 5e14 pfu or at least 1e15 pfu or atleast 3e15 pfu.

The homogeneous phage populations may be used for producing homogeneous,high yield populations of ssDNA packaged inside of phages. The ssDNA maybe extracted by lysing the phages. As for a phage length, a length ofssDNA (or a number of bases in it) may be determined by selecting alength (and/or a number of base pairs) of the first artificial plasmid.In general, when all other conditions are the same, a longer (havingmore base pairs) first artificial plasmid will result in a longer(having more bases) ssDNA, while a shorter (having less base pairs)first artificial plasmid will result in a shorter (having less bases)ssDNA. Due to the homogeneity of the phage populations, the producedssDNA may also have a high degree of homogeneity, such as homogeneity insize.

The produced ssDNA may contain from 135 to 100,000 bases or from 135 to80,000 bases or from 135 to 60,000 bases or from 135 to 50,000 bases orfrom 135 to 40,000 bases or from 30,000 bases or from 135 to 20,000bases or from 135 to 15,000 bases or from 135 to 12,000 bases or from135 to 10,000 bases or any integer number or subrange within theseranges.

In some embodiments, the first artificial plasmid may be shorter than awildtype plasmid.

For example, in some embodiments, the produced ssDNA may contain from179 to 6400 bases or from 135 to 6300 bases or from 135 to 6200 bases orfrom 135 to 6100 bases or from 135 to 6000 bases or from 200 to 5000bases or from 200 to 4000 bases or from 200 to 3500 bases or from 200 to3000 bases or any integer number or subrange within these ranges.

In some embodiments, the first artificial plasmid may be longer than awildtype plasmid. For example, the first artificial plasmid may containfrom 6450 to 100,000 bases or from 6500 to 100000 bases or from 6500 to80,000 bases or from 6600 to 70,000 bases or from 6800 to 60,000 basesor from 6800 to 50,000 bases or from 7000 to 40000 bases or from 7000 to35000 bases or from 7000 to 30000 bases or from 7000 to 20000 bases orfrom 7000 to 15000 bases or any integer number or subrange within theseranges.

The present method may allow producing homogeneous phage populationswith a high phage count without a need for purifying a desired portionof a phage population, i.e. a portion, which has a desired phage lengthvalue, from undesired phage portions, i.e. phage portions, which are notsubstantially close to the desired phage length value. For example, thepresent method does not include enriching desired portion of a phagepopulation by PEG precipitation and column separation. Nevertheless, insome embodiments, the method may include purification of the producedphage population. For example, the present method may involvepurification to remove unwanted and/or contaminating proteins from thephage population. Such purification may be performed, for example, usingcesium chloride gradient ultracentrifugation and column separation.

Homogeneous phage populations may be used for a number of applications.

In some embodiments, homogeneous phage populations may be used as adelivery vehicle for delivery an active agent, which may be attached toand/or conjugated with phages of the phage population, to a particulararea of a body of a subject, such as a mammal, e.g. a human. The activeagent may be, for example, a therapeutic agent and/or an imaging agent.Accordingly, the homogeneous phage population may be used in atherapeutic and/or imaging method, which may involve administering thehomogeneous phage population to a subject, such as a mammal, e.g. ahuman being. The homogeneous phage population may be, for example,administered topically, enterally or parenterally. For example, thehomogeneous phage population may be swallowed, injected or inhaled.

In some embodiments, the homogeneous phage population may beadministered intravascularly. In some embodiments, the homogeneous phagepopulation may be administered intracranially. In some embodiments, thehomogeneous phage population may be administered intradermally,subcutaneously or via intramuscular injections.

In certain embodiments, the homogeneous phage population may beadministered, for example, intravascularly or intracranially, fortreating and/or imaging a tumor, such as a central nervous system (CNS)tumor. The homogeneous phage population may be useful for treating bothadult and pediatric CNS tumors. CNS tumors include tumors ofneuroepithelial tissue, such as astrocytic tumors, oligodendroglialtumors, oligoastrocytic tumors, ependymal tumors, choroid plexus tumors,astroblastoma, neuronal and mixed neuronal-glial tumors, tumors of thepineal region, embryonal tumors; tumors of cranial and paraspinalnerves; tumors of the meninges, such as tumors of meningothelial cells,sesenchymal tumors, primary melanocytic lesions, haemangioblastoma;tumors of the haematopoietic system; germ cell tumors; tumors of thesellar region.

A homogeneous phage population may be a part of a composition, which maybe a pharmaceutically acceptable composition. The composition mayfurther include an acceptable carrier, which may be, for example, asterile buffer, such as PBS buffer or saline buffer, or purified water,such as Mille-Q water. The composition may also include one or more cellmedia components, such as blood derived serum, and/or one or morecomponents, such as enzymes, gelatin and amino acids.

A phage concentration in the composition may vary. For example, a phageconcentration may be from 1 e10 to 1e15 pfu/ml or from 1 e11 to 1e15pfu/ml or 1e12 to 1e15 pfu/ml.

In some embodiments, the homogeneous phage population may be implantedin a body of a subject, such as a mammal, e.g. a human. In certainembodiments, the homogeneous phage population may be a part of animplant, which may be, for example, one or more of a scaffold, ahydrogel and a nanostructure. The implant may act as a delivery and/ordetector device. Courchesen et al, Advanced Materials, 2014, 26(11),3398-3404), and Chen et al, Advanced Materials, 2014, 26(30), 5101-5107demonstrate method of crosslinking phage solutions to create viralscaffolding for various materials. Flynn et al, Acta Materialia, 2003,51(19), 5867-5880, Huang et al, Nano Lett., 2005, 5(7), 1429-1434, andHess et al, ACS Synth. Biol., 2013, 2(9), 490-496 demonstrate somemethods of assembling phage in structures, such as nanostructures.Implant type viral hydrogels may be produced via a variety of buildingmethods, such as glutaraldehyde crosslinking, layer-by-layer deposition,nanoparticle linkers, covalent bonding, DNA/sortase binding,lyophilization, photodynamic or UV crosslinking. FIG. 12 illustrates,for example, multiphage scaffolds and/or nanostructures.

In some embodiments, an active agent may be an imaging agent. An imagingagent may be an agent that is capable to produce a signal, the detectionof which may provide an image of an area of the subject's body, such asa tumor area. For example, an imaging agent may be a fluorescent imagingagent, such as a fluorescent label; a magnetic imaging agent, such as amagnetic nanoparticle; a radioactive imaging agent, such as aradioactive label; an infrared imaging agent; a photoluminescent imagingagent. One example of an imaging agent may be carbon nanostructure, suchas a carbon nanotube, which may be for example, a single wall carbonnanotube. Nanostructure imaging agents conjugated to phages aredisclosed, for example, in US 20130230464, which is incorporated hereinin its entirety.

In some embodiments, a signal from an imaging agent may be used to trackand diagnose cells or masses in a body of a subject, such as a human.For example, a signal from an imaging agent may be used to track anddiagnose cells or masses in a brain and/or spinal cord of a subject,such as a human. A signal from an imaging agent may be also used tovisualize a treatment over time.

In some embodiments, an active agent may be an infrared imaging agent,such as a carbon nanostructure, such as a carbon nanotube, which may bea carbon nanotube, such as a single wall carbon nanotube. In someembodiments, the imaging agent may be an organic or inorganic dye, whichmay emit in the near infrared range, which may be a range from 700 nm to2000 nm or from 800 nm to 1900 nm or from 900 nm to 1800 nm or from 800nm to 1750 nm or from 1100 nm to 1700 nm or value or subrange withinthese ranges. For example, a dye may be sodium yttrium fluoride dopedwith ytterbium and erbium. To detect an infrared signal from theinfrared imaging agent, after it has been administered to a subject,such as a human, together with the homogeneous phage population, aninfrared imaging system disclosed in Ghosh et al, PNAS 2014 111 (38)13948-13953 may be used. Such system may have imaging depths such as 10cm.

In some embodiments, an active agent may be a fluorescent imaging agent,such as, for example, Cy5.5 or Alexa-647. In some embodiments, afluorescent imaging agent may be conjugated directly to a phage coatprotein, such as pVI, pVII, pVIII or pIX. Yet in some embodiments, afluorescent imaging agent may be conjugated to another moiety, which isconjugated directly to or expressed by a coat protein of the phage. Forexample, a fluorescent imaging agent may be conjugated to chlorotoxin,which is directly conjugated to or expressed by pIII protein of thephage. Imaging using a fluorescent imaging signal may involve detectinga fluorescent signal from the label after it has been administered to asubject, such as a human, together with the homogeneous phagepopulation. For the detection, a multiphoton spectrometer or microscopemay be used.

In some embodiments, an active agent may be a therapeutic agent. Forexample, in some embodiments, a therapeutic agent may be an anticanceragent, such as a chemotherapy agent, e.g. doxorubicin. Use ofchemotherapy agents with phages is disclosed, for example, in Ghosh etal, ACS Synth Bio., 2012 1(12), 576-582. In some embodiments, atherapeutic agent may be a small molecule. Non-limiting examples oftherapeutic agents include siRNA or small DNA for cell-disruption,adjuvants for immunotherapy, nanoparticles such as gold nanoparticles,or theranostics including photodynamic therapy agents such as IR700. Ahost of agents may be conjugated to the phage via methods such as EDCchemistry, streptavidin-biotin strategies, zinc-finger sortasestrategies, metal chelating linkers such as DOTA-NHSester, thiolalkylation, maleimide modifications, and N-terminal transamination.Other amino acid handles may be used including cysteine residues fordi-sulfide bonds and tyrosine residue modifications. Peptide sequencesconferring affinity to molecules of interest (as determined by phagedisplay panning) may also be engineered to the coat proteins of thephage. (Bernard et al, Front Microbiol, 2014, 5(734), PMC4274979) (Hesset al, Bioconjug Chem., 2012, 23(7), 1478-1487)

In some embodiments, the homogeneous phage population may be apopulation of phages conjugated with a targeting moiety. The targetingmoiety may be such that it preferentially binds to a particular type ofcells compared to other cells. For example, the targeting moiety may bea tumor targeting moiety, i.e. a targeting moiety that bindspreferentially to tumor cells compared to other types of cells, e.g.normal cells. In some embodiments, the targeting moiety may bechlorotoxin. Chlorotoxin may selectively and/or preferentially bind tocertain types of tumors, such as gliomas and tumors of neuroectrodermalorigin such as medulloblastomas, neuroblastomas, melanomas, primitiveneuroectodermal tumora (PNETS), and small cell lung carcinoma.

In some embodiments, homogeneous phage populations, with or without anactive agent, may be used for treating a plaque caused by or associatedwith a neurogenerative disease, such as an Alzheimer's disease and othertypes of dementia; Parkinson's disease (PD) and PD-related disorders;prion disease; motor neurone diseases; Huntington's disease,spinocerebellar ataxia; spinal muscular atrophy.

The present inventors also developed a complex comprising a phage, suchas a filamentous phage, and chlorotoxin, which may be conjugated to thephage or expressed by the phage during phage assembly. For example,chlorotoxin may be conjugated to or expressed by the phage's coatprotein, such as pIII protein, or expressed by the phage's coat protein,such as pIII protein, during the phage assembly through engineeringphage sequence. In some embodiments, the phage may be a wildtype phage.In some embodiments, the phage may be an engineered phage, such as aphage, which is a part of a homogeneous phage population discussedabove. In some embodiments, chlorotoxin may be a labeled chlorotoxin,i.e. chlorotoxin with one or more labels attached to and/or conjugatedwith it. The label may be, for example, a radioactive label or afluorescent label. One example of example of labeled chlorotoxin may beCy5.5 labeled chlorotoxin, which is a combination of chlorotoxin and afluorescent material Cy5.5.

Chlorotoxin-phage may be prepared by incorporating a chlorotoxinencoding sequence, such as SEQ ID No. 1, into one of phage coat proteingenes of a plasmid, so that the chlorotoxin will be expressed by thecorresponding coat protein. For example, the chlorotoxin encodingsequence may be incorporated into pIII gene of M13 bacteriophage andthus, chlorotoxin may be expressed by pIII protein. A phage forexpressing chlorotoxin may be any phage whose genome may be edited. Forexample, a phage for expressing chlorotoxin may be any of the phagesdisclosed above. For example, the phage may be one of filamentous orfilamentous phages disclosed above. In some embodiments, a phage forexpressing chlorotoxin may be T4 phage, T7 phage, tobacco mosaic virusor potato virus.

For the wildtype phage, the chlorotoxin encoding sequence may beincorporated into a phage coat protein gene, such as pIII gene, of awildtype helper phage to produce a helper phage construct containing thechlorotoxin encoding sequence.

Yet for the engineered phage, the chlorotoxin encoding sequence may beincorporated into a phage coat protein gene, such as pIII gene, of thefirst or second artificial plasmid discussed above. For example, in someembodiments, for the engineered phage, which is a part of the disclosedabove homogeneous phage populations, the chlorotoxin encoding sequencemay be incorporated into the modified helper phage plasmid, which actsas the second artificial plasmid, to produce a modified helper phageconstruct containing the chlorotoxin encoding sequence.

The plasmid containing the chlorotoxin encoding sequence may betransformed through a bacterial strain, such as the ones discussedabove. For the wildtype phage, the plasmid containing the chlorotoxinencoding sequence may be transformed by itself without other plasmids.For the engineered phage, the plasmid containing the chlorotoxinencoding sequence may be co-transformed with the other plasmid disclosedabove. For example, when the second artificial plasmid contains thechlorotoxin encoding sequence it may be co-transformed with the firstartificial plasmid. When the first artificial plasmid contains thechlorotoxin encoding sequence it may be co-transformed with the secondartificial plasmid.

The products of transformation and or co-transformation may be amplifiedas discussed above and also in examples below.

In some embodiments, chlorotoxin-phages may be further conjugated withan active agent, which may be an imaging agent and/or a therapeuticagent. For example, when chlorotoxin is expressed by the pIII protein ofthe phage, an imaging agent, such as a fluorescent label or a carbonnanostructure may be conjugated or attached to the pVIII protein of thephage. For example, a fluorescent label, such as AlexaFlour may beconjugated with the pVIII protein through a reaction with side chains oflysine groups. For attaching a carbon nanostructure, such as a singlewall carbon nanotube, DSPH or other carbon nanotube complexing peptidemay be conjugated with or expressed by the pVIII protein of the phage.This may be accomplished by incorporating a sequence for DSPH or asequence for a carbon nanotube complexing peptide at the pVIII gene of arespective helper phage sequence before its transformation into abacterial strain.

The homogeneous phage populations and/or CTX-phage populations may havean ability to penetrate a blood brain barrier in subject such as ahuman, see FIG. 11 as well as additional discussion in Example below.For example, phage populations having lengths of no more than 900 nm orno more than 800 nm or no more than 700 nm or no more than 600 nm or nomore than 500 nm or nor more than 400 nm or no more than 300 nm or nomore than 200 nm or no more than 150 nm or no more than 100 nm may havean ability to penetrate a blood brain barrier when administeredintravascularly.

The homogeneous phage populations may be also used in a method of massproducing ssDNA of varying lengths/sequences. For example, homogenousphage populations produced with desired length or sequence of ssDNApackaged within may be produced at titers of at least 1e14 or at least1e15 or at least 3e15 or at least 1e16 phage from 10 L growths. Thesephage may be lysed and high yields of ssDNA extracted for use.

The present application also provides a kit for making a homogeneousphage population, which may include the first artificial plasmiddisclosed above; and the second artificial plasmid disclosed above. Inaddition, the kit may include a bacterial strain, which may be used forto con-transforming the first and the second artificial plasmids into itto produce a homogeneous phage population.

Embodiments described herein are further illustrated by, though in noway limited to, the following working examples.

EXAMPLE M13 Phage Assembly System for Optimal Blood Trafficking, TumorPenetration, and Passage Across the Blood Brain Barrier

This example relates to the functionality of M13 bacteriophage as anengineerable biomaterial for medical imaging and therapy applicationsparticularly in the case of tumor diagnosis. M13 phages of engineeredsizes (less than <50 nm to above a micron in range), termed ‘inho’phages were successfully assembled. M13 phages displaying chlorotoxinpeptide on the tail capsid of M13 were also successfully assembled. Theconstruction of shorter phage may improve on the extravasation and bloodtrafficking of M13 probe systems while retaining its multi-functionalcapsid proteins which may allow simultaneous targeting, detecting,and/or delivery of various agents, such as active agent, e.g. atherapeutic agent and/or an imaging agent, to a desired body part, suchas a cancerous tissue or a diseased tissue. Construction of phageexpressing chlorotoxin (CTX) as well as its reduced size may allow forthe passage of M13 probe systems across the blood-brain barrier (BBB)and the potential targeting to adult and pediatric brain tumors.

Technical Description

M13 filamentous bacteriophage is composed of a circular single strandedDNA (ssDNA) encapsulated by the major coat protein p8 and minor capproteins p3, p6, p7, p9 [1, see section “References” below]. Theseproteins can be engineered to display or attach various targetingsequences and nanoparticles or drug molecules—effectively creating aphage shuttle that carry imaging or therapy agents to specificallytargeted cancer cells [2]. In our system, we can control the length (orthe number of p8 coat) of the M13 phage by manipulating the length ofthe circular ssDNA plasmid packaged during the M13 assembly process. Aset of plasmids (termed “inho”) that generate package-able viral genomeswith desired sizes (i.e. 100s to 1000s base-pairs), which may be muchsmaller than the 6407 nucleotides observed in wildtype M13 (−880 nm inlength), was created. These package-able genomes may be of varyinglengths and contain the phage packaging signal and the f1 origin andtermination of replication but none of the phage protein genes. Ineffect, construct with the inserts produces ssDNA of a given length thatsignal to be packaged (FIG. 1). To produce minimally sized ssDNA, thepackaging signal region may also be removed and the packaging genomeretaining the f1 origin may be enough to begin assembly.

In the absence of M13 protein coding in the packaged genomes, a secondplasmid (RM13-f1) which expresses all essential phage assemblycomponents but itself lacks the packaging signal and f1 replicationorigin, was constructed. Only in the presence of both of these twoplasmids is phage production of the given size observed (FIG. 2), whereinho plasmids are packaged by the proteins translated from theunpackaged large RM13-f1 plasmid.

Co-transformation of an inho and the protein construct into a competentbacterial strain (in the present case XL-1 or DH5a) and overnightamplification of the co-transformed colony provide sufficient number ofphage for analysis. Using these two plasmids in concert, one can thenproceed to purification of the extruded phage-inhos for biomedicalapplications. In example, inho1960, inho475, and inho285 with RM13-f1gives us phage of 280 nm, 100 nm, and 50 nm in length (FIG. 3).Additionally, the RM13-f1 plasmid is open to any manipulations, such asthe addition of chlorotoxin display that may further improve on thespecificity of phage delivery of an active agent, such as an imagingagent and/or a therapeutic agent.

Chlorotoxin (CTX) is a 36 amino-acid (3995.8 Da) peptide derived fromthe venom of the Leiurus quinquestriatus scorpion. Recent studies haveshown that the CTX peptide selectively binds to and invades malignantgliomas (GBM) and tumors of neuroectrodermal origin such asmedulloblastomas, neuroblastomas, melanomas, PNETS, and small cell lungcarcinoma [3]. Additionally, native CTX as well as fluorophoreconjugated CTX tumor paint display the ability to cross the blood-brainbarrier (BBB) in both animals and humans with brain tumors, which maymake it an ideal trafficking peptide for nanoprobes designed for braintumor diagnosis and theragnosis. In order to harness these uniqueproperties to the M13 probe system, a phage clone that encodes for CTXon the p3 capping protein of the M13 phage (both wildtype and inho orshort phage) was developed. The 36 amino acid sequence of the CTXpeptide is incorporated into the gene for p3 as illustrated in FIG. 4.

The CTX peptide is highly positive in charge, an attribute which may bepart of the reason for its ability to cross the BBB and target to tumorareas with extracellular matrix which is particularly negative in charge[3]. When suspended with negatively charged citrate stabilized gold/Aunanoparticles, CTX phage electrostatically interacts with thenanoparticles at its CTX-p3 end (FIG. 5).

Chlorotoxin also has a distinct in vitro cellular localization anduptake pattern in human glioma versus normal cells, where it localizesnear the golgi apparatus in glioma cells . Using the CTX phageparticles, in vitro cellular uptake and colocalization of phageparticles to the Golgi apparatus in human adult U87MG glioma cells(FIGS. 6A & B) as well as human pediatric D458 medulloblastoma cells(FIGS. 6C & D) as early as 6 hrs following incubation of cells CTX phagewith continued accumulation at 24 hrs, were shown, demonstrating theversatility of the CTX phage to target multiple types of brain tumors.

In both immunocompromised (FIG. 7) and immunocompetent (FIG. 8)intracranial orthotopic mouse models of GBM, targeting to the tumor siteby CTX phage delivered intravascularly via tail vein injection wasobserved. Accumulation of CTX phage on the tumor surface is observableon a level much greater than wild-type control phage as demonstrated inFIGS. 7 and 8.

It had been previously demonstrated that the M13 bacteriophage can beused as a molecular shuttle for the delivery of various therapymolecules and imaging agents (i.e. single walled nanotubes, SWNT) to thesite of tumors. These molecular carriers have been most effective incombination with the second window near infrared (NIRII) imaging systemwhich allows for deep signal penetration through tissue usingnon-radioactive, inexpensive imaging machinery, see e.g. US 20170017069,Ghosh et al, PNAS 2014 111 (38) 13948-13953. Illustrated here (FIG. 9)is the NIRII brain tumor signals from the dual phage SWNT probe and NIRimaging system which may give surgeons the information needed to removetumors at the sub-millimeter size level—and those tumors that were notdetectable by the naked eye.

These preliminary data may reveal successful localization to gliomamasses from long SWNT complexed CTX phage (880 nm) as well as short SWNTcomplexed CTX phage (300 nm).

Advantages and Improvements over Existing Methods

Firstly, filamentous phages of shorter lengths by constructing a set ofsmall viral ssDNA that are packaged by M13 capsid proteins (thesesmaller phage retains the M13 major and minor coat proteins) wereengineered. Now with the ability to control the aspect ratio of theserigid, rod-like phages one can further improve on M13 basedcancer/disease detection or panning by optimizing for phage bloodcirculation and tumor extravasation. Secondly, collection by cloning forchlorotoxin display on the tail p3 capsid protein of M13 has been added.CTX may induce passage across the BBB and target glioma tumors in vivo.Expression of CTX on M13 may allow capitalizing on its strong affinityfor tumors of neuroectodermal origin, increasing its usage for thedetection and delivery of novel therapies to treat adult and pediatricbrain tumors with the potential to expand the platform for use in otherdiseases of the central nervous system.

Two Plasmid Phage Assembly Method

Preceding work by Specthrie et al. created a plasmid that generates aminimal packaging genome and produced a mixed population of full lengthphage and smaller phage that are 50 nm in length (though only 1-3% bymass of total population) [4, 5, 6]. The present work differs fromSpecthrie et al. in two ways. The first is to construct a set ofplasmids that generate package-able genomes with any needed size. Thesecond is to generate populations of a shorter size by removing thehelper phage using a plasmid that contains all phage proteins but isunable to package its DNA. In this way, homogenous batches of phage withlengths below 50 nm and above (length ranges in the microns alsopossible) can be produced. FIG. 22 provides evidence of a high degree ofhomogeneity in length for inho phages. In particular, FIG. 22 presents ahistogram distribution of inho phage sizes as measured from AFM imaging:for inho475 and inho1960 phages, about 90% of all measured lengths fallwithin the base size bin

Tumor or Disease Targeting In Vivo

The Biomolecular Materials Lab (Belcher Group) at the Koch Institute atMIT has previously demonstrated that targeted M13 bacteriophageconjugated with fluorescent materials may be used to perform in vivomolecular imaging of tumors [1]. However, this method may be limited byinefficient extravasation of the probe into vasculature due to thelength of the phage (880 nm). To improve tumor penetration of phage, asystem to control and shorten the length of phage while maintaining itsmulti-functional capsid has been engineered. The geometry and size mayplay a significant role in the transport, bio-distribution, andinternalization of nanoparticles [7, 8]. Upon systemic injection,nanoparticles must: 1) Evade uptake by circulating immune macrophages;2) Achieve marginalization and escape the circulation to reach bloodvessel walls; 3) Extravasate into the tumor interstitium; and finally,4) bind to or be internalized by cancer cells [9]. Though sphericalparticles have been the norm in nanomedicine research, non-sphericalnanoparticles (i.e. rods, chains, ellipsoids) may be more effective inthese areas [10]. Not only are chain or rod-like structures more likelyto avoid internalization by macrophages, their shape subjects them tocertain torque and tumbling motion that increases contact with thevessel walls. Furthermore, oblong shaped particles are more likely toform multivalent occurances essential for targeting—in the case of thefilamentous bacteriophage, avidity of binding can be highly enhanced bythe display of materials on all 2700 copies of the body p8 protein. Onthe other hand, size considerations must be made to accommodate for thehigh interstitial flow pressure typical of tumor masses. Due to theincreased leakiness of tumor vessels and reduced lymphatic drainage,extravasation and delivery of nanoparticles to tumor tissues can beachieved via enhanced permeation and retention (EPR) effects, which istypically seen with smaller particles <100 nm in diameter.

Delivery Past the Blood Brain Barrier

The permeability of the BBB to nanoparticles is affected by a number offactors including size, charge, and surface chemistry of the particles.M13 bacteriophage has been postulated to be able to cross the BBB. Whilelong in length, the structure of M13 is extremely narrow in diameter(−5-6 nm) and easily falls under the hydrodynamic size limit ofparticles that freely pass the BBB [3, 11]. Engineering the M13 platformto enable easy passage across the BBB may be particularly advantageousin concert with deep tissue imaging technologies. The brain is an organwhich is especially difficult to image in depth-imaging the CTX-phageprobes may be performed using the near infrared imaging system which hasdepths up to 10 cm. Ghosh et al, PNAS 2014 111 (38) 13948-1395; Dang etal, PNAS, 2016, 113(19), 5179-5184.

Given the capabilities of the CTX phage to cross across the BBB andinternalize to tumor cells, one may utilize the system for simultaneousdelivery of imaging agent and gene or chemo-therapies. Combining theinho and CTX designs may allow one to further achieve penetration intotumors, such as glioma and medulloblastoma tumors. Small sizesachievable by inho phage (less than <50 nm) may allow penetration ofinho phage across intact/heathly BBB of non-tumor bearing mice.Additionally, due to the high pay load possible with each inho shuttlevs. single molecules such as tumor paint, the M13 shuttles may beparticularly important as a means of increasing distribution of drugspast the BBB and for reducing nonspecific systemic effects. Furthermore,the multifunctional M13 shuttle may play a significant role in theimaging detection, drug treatment, and surgical removal of brain tumorswhich require extremely precise handling for patient safety.

In addition, M13 phage has been studied as a means of reversing theformation of plaques derived from amyloid-like structures in the brainrelated to Alzheimer, Parkinson and other neurodegenerative diseases[12]. The P3 capsid protein was identified as a major agent withcurative potential and was engineered as a recombinant bivalent G3Pmolecule called General Amyloid Interactive Motif (GAIM) by NeuroPhagePharmaceuticals or more recently renamed Proclara BioSciences [13]. Thissmall motif was mainly developed in order to overcome the hurdle ofdelivering M13 bacteriophage across the BBB while retaining the abilityof the bacteriophage to bind to beta amyloid protein in the brain. Theeffectiveness of M13 phage to clear amyloid plaques is not completelycaptured with GAIM, which suggests that CTX phage as well as inho-phagemay be a more powerful alternative to targeting plaques in the brain.

EXPERIMENTAL

Cloning CTX-phage. Helper phage template M13KE was used as the basis ofthe addition of CTX peptide display at the c-terminal end of the p3capsid protein (see FIG. 4 for insertion sequence).

Construct Production Steps: Display of CTX insert was achieved throughrestriction enzyme sites at the p3 region of the New England Biolabs(NEB) M13KE vector. The 108 base-pair CTX insertion and oligos designedwith Acc65I and EagI enzyme cut sites were purchased through IDT. TheCTX template was used in a PCR reaction (KAPA HiFi Kit) to amplify CTXinserts with the previously mentioned enzyme cut sites and purifiedthrough a 1.2% agarose gel run and extraction (QIAquick Gel Extraction).Both the host vector M13KE and the CTX PCR product were digestedovernight at 37° C. with Acc65I and EagI NEB enzymes in Buffer 3.1.Dephosphorylation of the host vector post digestion was conducted at 37°C. for 1 hour with 2.5 μL of rSAP enzyme to reduce recircularizationevents in the host vector. The final CTX and M13KE digestion productswere further purified through agarose gel run and extraction. T4ligation with the prepped CTX insert and M13KE vector was performedovernight at 16° C.

1-2 μL of the ligation products were transformed according tomanufacturer's instructions for competent cells (i.e. XL-1 Blue),prepared in 3 ml of agar top and 100 μL of overnight bacterial culture(XL-1 Blue), and incubated on IPTG-X-Gal agar plates overnight at 37° C.Phage plaques from the plates were picked, grown overnight, and preppedfor sequencing (QIAprep). Finally, full sequencing of the purifiedplasmids were verified for desired final M13KE-CTX construct. A map ofCTX insertion is shown in FIG. 13.

Cloning RM-13-f1 Constructs. Commercially available helper phagetemplates M13KE, M13K07, and R408 were used to create RM13-f1 constructswhere the intergenic region is reassembled to disrupt the origin forpackageable ssDNA replication (f1 site in particular was removed, FIG.14 presents a part of the removed sequence). Kanamycin resistance sitewas added to the constructs for identification purposes and ColE1(p15a-ori) plasmid replication site is included to achieve optimal copynumbers during E. coli growth. Additional functionalization (i.e. CTX onp3, DSPH or CNT complexing peptide display on p8, HIS-tag on p3,separation of overlapping capsid sequences) of the phage is easily alsoachieved as described below.

Construct Production Steps: Plasmid cloning by PCR with restrictionenzyme or gibson master assembly techniques were used to createconstructs from vector fragments of interest or to add insertionsimportant to functionalizing the capsid proteins. All oligos and smallDNA inserts were purchased through IDT. PCR reactions were performed(KAPA HiFi Kit) to amplify inserts and vectors with/out the enzyme cutsites or gibson overlapping overhangs. PCR products were purifiedthrough a 1.2% agarose gel run and extraction (QIAquick Gel Extraction)or DNA spin columns. Standard ligation or Gibson reactions wereconducted as appropriate. 1-2 μL of the ligation or Gibson products weretransformed according to manufacturer's instructions for competent cells(i.e. XL-1, DH5alpha) and incubated on antibiotic kanamycin agar platesovernight at 37° C. Bacterial colonies from the kanamycin plates werepicked, grown overnight, and prepped for sequencing (QIAprep). Finally,full sequencing of the purified plasmids were verified for desired finalRM13-f1 construct. FIG. 15 is a map of RM13-f1, RM13-f1 with p8modification (DDAH), with p3 modification (HIS6 (SEQ ID NO: 7)), with p8and p3 modification (DSPH & CTX).

Cloning Inho Constructs. Inho construct were assembled via theincorporation of the f-1 origin of replication (modified for origin ofssDNA replication only), f-1 termination site (a repeat of the f-1origin with deletions that render ineffective the nicking of the plasmidfor ssDNA production), and the packaging signal (PS). Ampicillinresistance site was added to the constructs for identification purposesand ColE1 plasmid replication site is included to achieve optimal copynumbers during E. coli growth. Addition and deletion of base-pairs inthe ssDNA region (as demonstrated in the case of inho1960, inho475 etc)may be easily achieved via the cloning procedures described. FIG. 16shows sequences of f1 replication modified for origin only (SEQ ID No.3), termination only (SEQ ID No. 4 and SEQ ID No. 5), and the packagingsignal as found in M13-ori (SEQ ID No. 6), while FIG. 17 identifiessections of ssDNA, which may be manipulated by for example, addingand/or replacing and/or deleting bases therein.

Construct Production Steps: Plasmid cloning by PCR with restrictionenzyme or gibson master assembly techniques were used to createconstructs from fragments of interest or to add insertions important tossDNA packaging and to change the length of the inho-phage. Insertionssuch as EGFP and GLuc sequences were used to test the efficacy of inhoconstructs with different ssDNA production region (See FIG. 18). Alloligos and small DNA inserts were purchased through IDT. PCR reactionswere performed (KAPA HiFi Kit) to amplify inserts and vectors with/outthe enzyme cut sites or gibson overlapping overhangs. PCR products werepurified through a 1.2% agarose gel run and extraction (QIAquick GelExtraction) or DNA spin columns.

Standard ligation or Gibson reactions with inserts or fragments wereconducted as appropriate. 1-2 μL of the ligation or Gibson products weretransformed according to manufacturer's instructions for competent cellsand incubated on ampicillin agar plates overnight at 37° C. Bacterialcolonies from the ampicillin plates were picked, grown overnight, andprepped for sequencing. Full sequencing of the purified plasmids wereverified for desired final inho construct.

Amplifying Inho-Phage

Phage Plates: 50 ng each of inho construct of desired base pair lengthand RM13-f1 with desired capsid proteins, such as e.g. CTX and/or DSPH,sequences are co-transformed through heat shock in tetracyclineresistant bacterial strain (i.e. XL-1 Blue) and grown SOC Media. The SOCgrowth is spread over kanamycin+ampicillin agar plates and incubatedovernight at 37° C. Resulting bacterial colonies are stored up to 3months for amplication of phage.

Amplification:

1. Sterile LB Media is prepared with tetracycline, kanamycin, andampicillin at working concentrations of 10 μg/mL, 50 μg/mL, and 100μg/mL respectively.

2. Bacterial colony picked from plate is grown overnight in LB at 37° C.and 225 rpm.

3. Resulting bacterial culture is spun for 30 min at 8000 rpm to removeE. coli bacteria.

4. The supernatant containing the phage product is stored overnight at4° C. with 10% PEG/NaCl to precipitate out the inho-phage particles.

5. The phage media is centrifuged again for 40 min at 8000 rpm to pelletout the phage.

6. Resuspend the pellet in sterile buffer or milliQ water.

Confirming Phage Identity:

1. Samples of the resuspend phage were denatured by heat and SDS and runon TBE gels to identify the packaged ssDNA (See FIG. 20)

2. Samples of the resuspended phage were denatured by heat and SDS andrun on Nu-PAGE gels to check for the presence of p3 capsid protein viaWestern Blot (See FIG. 21).

3. Diluted samples of phage were imaged via AFM and TEM.

Amplifying CTX-Phage

Phage Plates:

50 ng of the CTX construct was transformed according to manufacturer'sinstructions for tetracycline resistant, competent cells (i.e. XL-1Blue), prepared in 3 ml of agar top and 100 μL of overnight bacterialculture (XL-1 Blue), and incubated on IPTG-X-Gal agar plates overnightat 37° C. Resulting phage plaques are stored up to 3 months foramplication of phage.

Amplification:

1. Sterile LB Media is prepared with tetracycline at workingconcentrations of 10 μg/mL.

2. Phage plaque picked from plate is grown overnight in LB at 37° C. and225 rpm.

3. Resulting bacterial culture is spun for 30 min at 8000 rpm to removeE. coli bacteria.

4. The supernatant containing the phage product is stored overnight at4° C. with 2.5% PEG/NaCl to precipitate out the inho-phage particles.

5. The phage media is centrifuged again for 40 min at 8000 rpm to pelletout the phage.

6. Resuspend the pellet in sterile buffer or milliQ water.

Confirming Phage Production:

1. Samples of the resuspended phage were denatured by heat and SDS andrun on Nu-PAGE gels to check for the presence of p3 capsid protein viaWestern Blot.

2. Diluted samples of phage were imaged via AFM and TEM.

Phage Purification Options:

1. Repeat PEG/NaCl precipitation.

2. Digest with DNase Ito reduce DNA debris.

3. Run dialysis against buffer of choice up to 72 hrs with at least 3refills

4. Gradient cesium chloride ultracentrifugation at 150,000 g for 4-8 hrsat 4° C., expecting white film of phage near the 1.4 density layer.

Complexation of Phage with CNT and Dyes

1. CNT/phage complexes were fabricated as previously described inpublished work, Dang et al. Nature Nanotechnology 6, 377-384 (2011) withp8 coat protein insert sequence DSPHTELP selected for SWNT binding andpH control allowing for efficient dispersion of the complex.

2. Fluorescent dyes from the AlexaFlour NHS Ester line where picked forlabeling of phage for imaging. The amine reactive chemistry of the dyesallows for easy labeling of side chains of lysine groups exposed by thep8 coat protein. Steps followed per manufacturer's instructions.

Labeled phage were processed under sterile conditions and dialyzedagainst buffer for 72 hrs before usage in vivo or in vitro studies.

In Vivo Imaging of Phage Complex in Mice

CNT complexed phage at varying concentrations [1e13, 6e13, 9e13 pfu/mL]and lengths [300, 880 nm] were prepared for 200 μL tail vein injectionsin NCR-NU male mice (homozygous) post intracranial situation of gliomatumors. The mice were monitored post injection at 16 hr, 48 hr, 72 hr onthe Belcher NIR-II Imager (setup at 808 nm laser, 2×1100 longpass,2×1300 longpass, 1 second exposure). NIR-II imaging as previouslydescribed by Ghosh et al, PNAS 2014 111 (38) 13948-13953. At 71 hrs endof trial, mice were sacrificed and dissected and the brain was imagedfor phage localization at the tumor site.

Chlorotoxin-expressing M13 Phage.

In Vitro Cellular Uptake Studies. Human U87MG glioblastoma cells weregrown on glass coverslips coated with Poly-L-lysine (Gibco) in DMEM cellmedia (Corning) supplemented with 10% fetal bovine serum (Sigma). HumanD458 medulloblastoma cells grown in suspension in DMEM/F12 media (Gibco)supplemented with 10% fetal bovine serum (Sigma), 1× Glutamax (Gibco),and 1× Penicillin/Streptomycin (Sigma). Cells were incubated at 37° C.with Cy5.5-conjugated CTX-expressing M13 phage for 6 hrs and 24 hrs.Coverslips with U87MG cells were washed three times with PBS beforefixing with paraformaldehyde and processed for immunofluorescence. D458cells were harvested into Eppendorf tubes, spun down at 1000 rpm X 5min, and washed three times with PBS before spinning onto glassmicroscope slides using a Shandon Cytospin (ThermoFisher).Immunofluorescence was performed on cells using Golgin 1 (Cell SignalingTechnologies) as a Golgi marker, and DAPI (ThermoFisher) as a DNA stainfor colocalization. Images were captured on an EVOS fluorescentmicroscope (Life Technologies) or a Nikon Eclipse 80i fluorescencemicroscope.

U87MG and GL261 Orthotopic Intracranial Tumor Implantation and In VivoTumor Monitoring. All animal experimentation was in adherence with theNational Institutes of Health (NIH) Guide for the Care and Use ofLaboratory Animals and received institutional approval. U87MG humanglioma cells were purchased through ATCC (ATCC No. HTB-14) andmaintained in DMEM media (Gibco) with 10% FBS (Hyclone). GL261 mouseglioma cells (Szatmari T et al, Cancer Sci, 2006, 97(6):546-553) weremaintained in DMEM (Gibco) with 10% FBS (Hyclone), 1× Glutamine (Gibco),and 1× Pen/Strep (Sigma). Cell lines were transduced with a lentiviralpLMP-GFP-Luc vector to allow for stable expression of GFP and fireflyluciferase prior to implantation. Six week old NCR nude (Taconic) orC57/BL6 male mice (Taconic) were used to generate intracranialorthotopic U87MG or GL261 gliomas, respectively. In brief, mice wereanesthetized using 2% isoflurane and their heads immobilized in astereotactic headframe using atraumatic ear bars. A burr hole was madeusing a steel drill bit (Plastics One, Roanoke, Va., USA) 1.4 mm rightof the sagittal and 1 mm anterior to the lambdoid suture. 105 gliomacells were stereotactically injected 3 mm deep from the dura mater intothe brain using a 33-gauge Hamilton syringe. Tumors were allowed to growfor 14 days prior to commencement of treatment. Intracranial tumorgrowth was monitored in vivo using bioluminescence IVIS® imaging(Xenogen, Almeda, Calif.) equipped with Livinglmage™ software (Xenogen).

Multiphoton Intravital Imaging of In Vivo Tumor Uptake byChlorotoxin-expressing M13 Phage Through A Cranial Window—To fashion acranial window, the skull was thinned away using a sterile stainlesssteel 2 mm diameter cylindrical drill bit attached to a high-speed handdrill until the underlying dura mater is exposed. Multiphoton imagingwas performed on an Olympus FV-1000MPE multiphoton microscope (OlympusAmericas, Waltham, Mass.) using a 25X, N.A 1.05 water objective.Excitation was achieved using a DeepSee Tai-sapphire femtosecond pulselaser (Spectro-Physics, Santa Clara, Calif.) at 840 nm. The emittedfluorescence was collected by PMTs with emission filters of 425/30 nmfor Collagen 1, 525/45 nm for GFP-labeled tumor cells and 668/20 nm forAlexa-647 nanoparticles. Collagen 1 was excited by second harmonicgeneration and emits as polarized light at half the excitationwavelength. Images were taken 24 hrs post-IV injection ofChlorotoxin-expressing M13 phage. All images were processed usingImageJ.

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Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention. All of thepublications, patent applications and patents cited in thisspecification are incorporated herein by reference in their entirety tothe same extent as if individually incorporated by reference.

What is claimed is:
 1. A homogeneous engineered filamentous phagepopulation, wherein at least 30% of filamentous phages in the phagepopulation have a length within 15% of a selected length value, which isgreater than the corresponding wild type filamentous phage length valueand up to 10 microns.
 2. The phage population of claim 1, wherein atleast 50% of filamentous phages of the phage population have a lengthwithin 15% of the selected length value.
 3. The phage population ofclaim 1, wherein at least 70% of filamentous phages of the phagepopulation have a length within 15% of the selected length value.
 4. Thephage population of claim 1, wherein at least 90% of filamentous phagesof the phage population have a length within 15% of the selected lengthvalue.
 5. The phage population of claim 1, wherein the selected lengthvalue is from 900 nm to 10 microns.
 6. The phage population of claim 1,wherein the selected length value is from 950 nm to 10 microns.
 7. Thephage population of claim 1, wherein the phage population has a count ofat least 1e13 pfu.
 8. The phage population of claim 1, wherein the phagepopulation has a count of at least 1e13 pfu.
 9. The phage population ofclaim 1, further comprising an active agent attached to filamentousphages of the population, wherein the active agent is selected from atherapeutic agent, an imaging agent and a combination thereof.
 10. Thephage population of claim 9, wherein the active agent is an imagingagent.
 11. The phage population of claim 10, wherein the imaging agentcomprises a carbon nanostructure.
 12. A therapeutic and/or imagingmethod comprising administering to a subject the homogenous engineeredfilamentous phage population of claim
 9. 13. The method of claim 12 fortreating and/or imaging a tumor.
 14. The method of claim 13, whereinfilamentous phages of the phage population comprise a tumor targetingmoiety.
 15. The method of claim 14, wherein the tumor targeting moietycomprises chlorotoxin.
 16. The method of claim 15, wherein thechlorotoxin is a fluorescently labeled chlorotoxin.
 17. The method ofclaim 15, wherein the tumor is selected from glioma, medulloblastoma,neuroblastoma, melanoma, primitive neuroectodermal tumor, and small celllung carcinoma.
 18. The method of claim 12, wherein the administeringcomprises intravascular administering.
 19. The method of claim 12,wherein the administering comprises implanting the homogeneousengineered filamentous phage population.
 20. The method of claim 19,wherein the said implanting is intracranial implanting.
 21. Apharmaceutical composition comprising the homogeneous engineeredfilamentous phage population of claim
 1. 22. An implant comprising thehomogeneous engineered filamentous phage population of claim
 1. 23. Theimplant of claim 22, which is one or more of a scaffold, a hydrogel or ananostructure.
 24. A phage comprising chlorotoxin expressed by a coatprotein of the phage, wherein the phage is an engineered phage, which isa part of a homogeneous engineered phage population, wherein at least30% of phages in the phage population have a length within 15% of aselected length value, which is greater than the corresponding wild typefilamentous phage length value and up to 10 microns.
 25. The phage ofclaim 24, wherein at least 50% of phages in the phage population have alength within 15% of the selected length value.
 26. The phage of claim24, wherein at least 70% of phages in the phage population have a lengthwithin 15% of the selected length value.
 27. The phage of claim 24,wherein at least 90% of phages in the phage population have a lengthwithin 15% of the selected length value.
 28. The phage of claim 24,further comprising an imaging agent.
 29. The phage of claim 28, whereinthe imaging agent comprises a carbon nanostructure.
 30. The phage ofclaim 24, wherein the chlorotoxin is a labeled chlorotoxin.
 31. Thephage of claim 31, wherein the chlorotoxin is a fluorescently labeledchlorotoxin.
 32. A pharmaceutical composition comprising the phage ofclaim
 24. 33. An implant comprising the phage of claim
 24. 34. Theimplant of claim 33, which is one or more of a scaffold, a hydrogel or ananostructure.
 35. A therapeutic and/or imaging method comprisingadministering to a subject the phage of claim
 23. 36. The method ofclaim 35 for treating and/or imaging a tumor.
 37. The method of claim36, wherein the tumor is selected from glioma, medulloblastoma,neuroblastoma, melanoma, primitive neuroectodermal tumor, and small celllung carcinoma.
 38. The method of claim 35, wherein said administeringis performed intravascularly.
 39. The method of claim 35, wherein saidadministering is performed intracranially.
 40. The method of claim 35,wherein said administering comprises implanting the phage.
 41. The phagepopulation of claim 1, which is a homogeneous engineered Ff phagepopulation.
 42. The phage population of claim 42, which is a homogeneousengineered M13 phage population.
 43. The phage of claim 24, wherein theengineered phage is an engineered filamentous phage.
 44. The phage ofclaim 43, wherein the engineered filamentous phage is an engineered Ffphage.
 45. The phage of claim 43, wherein the engineered filamentousphage is an engineered M13 phage.
 46. A kit comprising: a firstartificial plasmid comprising an f1 origin replication sequence; and asecond artificial plasmid, which does not comprise an f1 originreplication sequence, wherein the first and second artificial plasmidtogether contain sequences encoding a complete library of phage coat andassembly proteins.
 47. A method of making a homogeneous phage populationand/or a homogeneous ssDNA population, comprising: obtaining a firstartificial plasmid comprising an f1 origin replication sequence;obtaining a second artificial plasmid that does not comprise an f1origin replication sequence; and co-transforming the first artificialplasmid and the second artificial plasmid into a bacterial strain toproduce a homogeneous phage population and/or a homogeneous ssDNApopulation, wherein the first and second artificial plasmid togethercontain sequences encoding a complete library of phage coat and assemblyproteins.
 48. A homogeneous engineered filamentous phage populationprepared by the method of claim
 47. 49. A homogenous ssDNA populationprepared by the method of claim 47.